A method for rapid generation of competitive standard molecules for RT-PCR avoiding the problem of competitor/probe cross-reactions.

Abstract

The analysis of gene expression is a widespread issue in a growing number of fields such as molecular genetics, immunology, and medical diagnostics. The ideal method for mRNA detection should be fast, inexpensive, sensitive, and reliable. Well-elaborated standard methods such as Northern hybridization, Sl-mapping, and RNAse protection are useful and recommended, but only reverse transcription PCR (RT-PCR) gives the highest possible sensitivity required. For many issues it is necessary not only to detect a distinct mRNA but to compare changes in mRNA levels. The use of RT-PCR for such semiquantitative and quantitative approaches resolves problems attributable to the intrinsic property of PCR, that is, the variability in the number of copies of products synthesized during the process. Slight differences in reaction conditions can result in drastic differences in product yield. For many applications it is sufficient to compare mRNA levels for a set of probes relative to each other. Even in this case, some parameters still remain to be standardized, for example, it has been shown that the position of the probe in the heating block might be crucial for the product yield. Therefore, protocols commonly used for this purpose include the addition of equal amounts of competitor DNA fragments to the probes to be coamplified during PCR reaction. (1-3) As the competitor fragments are added in equal amounts they should yield equal amounts of PCR products. If the fragments derived from mRNA and the competitor are distinguishable in size, they can be separated easily by gel electrophoresis and visualized by ethidium bromide staining. Some requirements should be met for generation of such competitor frag